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JOURNAL OF BOTANY
Vol. VI June, 1919 No, 6
A STUDY OF PLASTIDS AND MITOCHONDRIA IN PRESSIA
W. C. Twiss
In this paper certain observations upon plastids and mitochondria are
recorded. In my opinion it would be premature as yet to formulate any
conclusions as to the fundamental significance of these bodies, though the
preparations I have obtained are clean-cut and definite.
In order to make clear the position assumed in regard to the structures
in question, it may be well to state, at the outset, that the term plastid will
be used to include not only the leuco-, chloro-, and chromoplasts, but also
the Anlagen for the same. The name mitochondria, on the other hand,
will be restricted to those granules which are not, in general, preserved by
the usual methods of fixation — those which, in other words, are dissolved
in acetic acid or in alcohol and are fixed by the use of osmic acid, formalin,
etc. The mitochondria, moreover, color more or less specifically with
various stains. I shall in general use Benda's term mitochondria, rather
than others that have been proposed, though the etymology of the word
implies a thread-like form not always present.
In cells prepared by what are known as the mitochondrial methods,
these bodies, by reason of their number and intense affinity for the dyes
become in many cases quite the most striking features of the protoplast.
The only reason that they were neglected by cytologists for so long a time is
the fact that they are dissolved by the processes commonly used to demon-
strate nuclear phenomena.
Interest in the granular constituents of the cytoplasm has greatly in-
creased in the last few years, though the idea of their importance is not a
new one. To Altmann, in 1886, is due the formulation of what is generally
known as the granular theory of protoplasmic structure. Hanstein, in
1882, had maintained that protoplasm is made up of minute granules, which
he termed microsomes, and a homogeneous fluid in which the microsomes
float. Altmann, using a special technique, consisting essentially of fixation
with osmic acid and potassium dichromate, was able to demonstrate the
presence in various tissue cells, as well as in the chromosomes, of numerous
granules to which he gave the name of bioblasts. These bodies he regarded
as possessing an independent existence, and to them he imputed the power
of growth and of multiplication by division. He also believed that they
[The Journal for May (6: 181-216) was issued June 20, 1919.]
218 W. C. TWISS
are transformed into the products of secretion such as fats, glycogen, pig-
ments, etc., in the cells. But Altmann, besides claiming for his bioplasts
the powers already noted, which are to a certain extent the same as those
believed by modern exponents of the mitochondrial theory to reside in the
mitochondria, held that the bioplasts are the morphological units of living
matter, constituting the essential elements of protoplasm. The difference,
in short, between Altmann's hypothesis and the more modern theories of
the mitochondria lies in the fact that the bioplasts were postulated to possess
an independence and autonomy, to quote Regaud, which the mitochondria
as cell organs are not thought to exhibit.
Benda, in 1898, having devised a more specific and definite method of
fixation and staining, may be regarded as the founder of the modern mito-
chondrial theory. He introduced the terms mitochondria and Chondrio-
miten, from /uros a thread, and x^ptov a grain. Chondriosome and
chondriocont, later introduced by Meves, have also come into use, the former
being used synonymously with mitochondria, and the latter being applied
by Meves to homogeneous threads. The collective term chondriome is also
Regaud ('n) suggests the possibility that the mitochondria "fix" and
"elaborate" the substances necessary for the functioning of the muscle
cells, such as glycogen, and that they also perform a similar function in the
case of the gland cells, such as the secretory cells of the kidney and of the
salivary glands. He says: "Les mitochondries sont les organites sur lesquels
se fixent les substances destinies au fonctionnement chemique de la cellule; ces
organites concentrent les substances fixSes, les Slaborent et les transforment en
produits de sScrStion, auxquels Us servent meme de supports, dont Us sont les
plastes." The mitochondria are "les agents de V intussusception elective,
c'est-a-dire, de V introduction dans la cellule des substances amen&es par le
Dubreuil ('13) believes that the mitochondria are responsible for the
production of fat in the cells, through a process of differentiation. Accord-
ing to Dubreuil, a lipoid vesicle is first formed, this process being followed
by the development of a fat droplet. The diagrams to illustrate this
process, showing the mitochondria becoming vesicular and forming both
hollow spheres and "hand-mirror-like" forms, exhibit a remarkable simi-
larity to the series of changes which Guilliermond and others show in their
illustrations of the production of plastids by the mitochondria in plant cells.
Van der Stricht ( '09) has presented some very suggestive studies upon
the genesis of yolk-spheres in the egg. He finds that the mitochondria are,
at first, confined to a region around the nucleus from which they migrate
outward and are gradually transformed into yolk-spheres.
Lewis and Lewis ('15) have employed a novel method for the study of
mitochondria with most interesting and suggestive results. Portions of the
living embryo of the chick were segregated under aseptic conditions and
cultivated in Locke's solution, in hanging drop cultures. In such cultures
A STUDY OF PLASTIDS AND MITOCHONDRIA 219
the mitochondria may be studied in the living, growing condition, fixed
while under observation and thus preserved as permanent preparations.
Experimental methods may be employed, by which the effect of different
stains and reagents may be observed directly. Such preparations, placed
in the electric incubator at a temperature of 39 to 40 C, show a beginning
of growth in from 10 to 20 hours. The new growth is attached to the cover
glass and is several cell layers in thickness at first, thinning out to a single
cell layer around the edges. Although conditions are necessarily somewhat
different from those of the normal somatic environment, especially as regards
circulation, and in the limited supply of oxygen since the cultures must be
hermetically sealed, the processes of growth and of mitosis seem to go on as
usual for a period of about three days. After this the process slows down,
growth ceases, and the cells finally die.
Mitochondria, conforming to the usual criteria, were found to be present
in all cells studied. Osmic acid vapor proved to be the best fixative, while
the vapors of acetic and other acids caused immediate and total disintegra-
tion of the mitochondria. The bodies appeared as threads and granules
of the most varied shapes and sizes, just as they have so often been described
in fixed preparations. They are not, however, constant in form or in size
under these conditions, but are constantly changing in appearance. They
are described as undergoing division, as fusing to form larger bodies, and
as disappearing and reappearing in the cells in a manner not accounted for.
During mitosis they are distributed regularly about the cell, so that they
are apportioned to the daughter cells in approximately equal numbers.
No connection of the mitochondria with the production of fats, such as
described by Dubreuil ('13), was noted in these preparations. In conclu-
sion, the authors state: "The mitochondria are extremely variable bodies,
which are continually moving and changing shape in the cytoplasm. They
appear to arise in the cytoplasm and to be used up by cellular activity.
They are, in all probability, bodies connected with the metabolic activity
of the cell."
Lewis and Robertson ('16) found that the above described method of
tissue culture was well adapted to the study of spermatogenesis in the
grasshopper, Chorthippus curtipennis. In the young spermatid the mito-
chondria were in the form of a granular Nebenkern. After certain internal
changes, the import of which was not clear, the Nebenkern was seen to divide
into half-spheres. These half-spheres then elongated to form granular sacs,
which, as the tail grew out, formed two irregular strands. These irregular
strands finally fused to form "two continuous threads of even width, ex-
tending from the centrosome body, or middle piece, almost to the end of the
The authors conclude that "it does not seem possible that bodies which
have to do only with the metabolic activities of the cell should undergo such
an exact behavior as shown, for instance, by the division of the Nebenketn
220 W. C. TWISS
into two equal parts and the development of these two sacs of mitochondria
into two long threads of mitochondria in the spermatozoon."
There has been a tendency, especially among the earlier observers of
mitochondria in plant cells, to ascribe to these bodies a nuclear origin.
A perusal of the literature clearly indicates that the work of Goldschmidt
( '04) is largely responsible for this, though the inception of the chromidial
hypothesis doubtless owes its origin, as Dobell ('09) states, to the work of
R. Hertwig, supplemented by that of Schaudinn, dealing with the occurrence
of such bodies in the protozoa.
Meves ('04), working with the tapetal cells in the anthers of Nymphaea
alba, is credited with having made the first observations of mitochondria
in plant cells. Meves shows two very clear-cut and beautifully drawn
figures, of which his description is as follows: " Enthalt sie lange, unregel-
massig gewundene, ziemlich dicke Faden, welche sich mil Eisenhamatoxylin
intensiv schwarz gejarbt haben. Diese Faden konnen nicht wohl etwas anderes
sein, als die von tierischen Zellen bekannten Chondromiten."
It is not so much the question of origin, whether sui generis or chromidial,
however, that has engaged the attention of later workers upon plant mito-
chondria, as that of their relation to other structures in the cell and of their
universality. Lewitsky and Pensa, working independently, have advanced
a contention which promises to furnish material for controversy for some
time to come.
Lewitsky ('io) studies the root-tip and stem-tip of Asparagus officinalis
treated according to the Benda method and stained with both the Benda
stain and haematoxylin. He finds mitochondria corresponding to those
described in animal cells, both in general appearance and in staining reac-
tion, with no evidence whatever of a nuclear origin. The mitochondria
appear short and rod-shaped, in the dermatogen; somewhat larger and with
a tendency to swell at the ends, in the second layer; still larger in the third
layer, while deeper in the assimilative tissue "dumb-bell" forms are seen,
"similar to those well known in division figures of the chloroplasts."
Next, still larger bodies are shown which appear as if they have come from
the separation of the two halves of the "dumb-bells." These are followed
by figures of the young chloroplasts, and finally by the mature bodies.
Meanwhile, the earlier forms of the mitochondria, combined with the
"division figures" of the intermediate regions, lead him to conclude that
the mitochondria are the Anlagen of the chloroplasts. Upon fixing some of
the same material in alcohol and acetic acid, Lewitsky found that the mito-
chondria were no longer to be seen in the cells, while the chloroplasts ap-
peared as usual. This is taken as evidence of a chemical as well as a mor-
phological transformation of the mitochondria, in producing the chloro-
Guilliermond ('n, '13, '14) has published a number of papers in which
he describes the mitochondria in all sorts of plants, his purpose being, on
A STUDY OF PLASTIDS AND MITOCHONDRIA 221
the whole, not so much to demonstrate the presence of mitochondria therein
as to substantiate his theory of their functional r61e. In addition to
portraying the same processes of development as those described by his
predecessors, Guilliermond seeks to show that the mitochondria of plant
cells possess the same "elaborative" functions that have been postulated for
animal mitochondria by Regaud and others. In a resume of the work upon
mitochondria, published in "La Revue generate de Botanique" in 1914, he
says: " Ces recherches demontrent surabondammentque les mitochondries sont
desplastes,c'est-d,-dire des organites quielaborentl&s produits de secrition. . . .
A la suite de ces recherches, la cellule apparait dSsormais avec un nouvel ele-
ment: le chondriome, dont la presence est aussi constante et joue un rdle aussi
essentiel que le noyau. . . . La decouverte des mitochondries transforme done
la cytologies Mottier ('18) has discussed the literature on the relations of
chondriosomes and plastids and it need not be further summarized here.
Although I was particularly concerned, in my own investigations, with
obtaining evidence as to the relationship between mitochondria and plastids,
the mitochondrial methods of fixing and staining were tested first upon
animal tissue. A number of preparations were made from the testes of the
grasshopper, Caloptenus femurrubrum. They were fixed with Benda's
solution, with Bensley's,and with Flemming's strong solution, and stained
in various ways, in order to compare the results obtained with different
Benda's method of fixing and staining gave by far the best results, the
mitochondria being well differentiated and shown in the characteristic
changes through which they pass, as described by Lewis and Robertson
('16) in their observations upon the behavior of the mitochondria during
spermatogenesis in the grasshopper, Chorthippus curtipennis. In my
preparations, Bensley's method gave unsatisfactory results in general
protoplasmic differentiation, though the mitochondria were well preserved
Benda's process, according to the formula given below, was therefore
used in most of my plant material.
Fixation. I. Benda's Flemming, 8 days.
(1 percent chromic acid, 15 cc,
2 percent osmic acid, 4 cc,
3 drops acetic acid.)
II. Wash in water, 1 hr.
III. Pyroligneous acid (rectified) and chromic acid 1 per-
cent, equal parts, 24 hrs.
IV. Bichromate of potassium, 2 percent, 24 hrs.
V. Wash in water, 24 hrs.
VI. Dehydrate and imbed. Cut 5 microns thick.
222 W. C. TWISS
Staining. VII. Iron alum, 4 percent, 24 hrs.
VIII. Rinse in distilled water.
IX. Alizarine sodium sulphonate, 24 hrs.
(1 to several cc, saturated alcoholic solution, in 80 to
100 cc. distilled water.)
X. Heat in crystal violet, 3 to 5 min. after vapor rises.
(3 percent alcoholic solution crystal violet and aniline
water, equal parts.)
XI. Rinse in distilled water.
XII. Destain in 15 percent acetic acid.
XIII. Wash in running water, 5 to 10 min.
XIV. Dry with filter paper. Dip in absolute alcohol.
XV. Bergamot oil, followed by xylol.
This stain gave a very beautiful result, the mitochondria being colored
a dark violet or blue, with a background of old rose. Only in exceptional
cases was the background too light to be satisfactory.
Regaud's method of fixing and staining was also employed, to some
extent, upon the plant tissues.
Fixation. I. Bichromate of potassium, 3 percent, 80 vols., and com-
mercial formalin, 20 vols., four days.
II. Bichromate of potassium, 3 percent, eight days.
III. Wash in water, 12 hrs.
IV. Dehydrate, imbed, and cut 5 microns thick.
Staining. V. Stain with iron-alum-haematoxylin (Heidenhain's
This is the formula as given by Guilliermond, who has used it in much
of his work. In my preparations it gave good results, at times, while again
the results might be very bad, possibly due to impurities in the formalin
The Benda fixation does just as well as a preparation for the iron-alum-
haematoxylin as for the crystal violet-alizarin stain, this combination being
In the root-tips of corn, of the " Canadian Early, Yellow Flint" variety,
fixed according to Regaud's formalin-bichromate method, the time being
shortened to four hours in the fixative (I), and eight hours in the bichromate
(II), the cytoplasm in the embryonic region appears gray and filled with
exceedingly numerous jet-black mitochondria, when stained with iron-
alum-haematoxylin. In this region the mitochondria are globular, ellip-
soid, or short rod-shaped. In the root-cap, next to the tip, they are similar,
gradually lengthening from short rod-shaped to elongated, filamentous
forms as one passes from the embryonic region toward the periphery of the
In the root-tip proper, passing back into the region of elongated cells in
A STUDY OF PLASTIDS AND MITOCHONDRIA 223
the plerome, a marked change occurs. The mitochondria now appear as
elongated, thread-like bodies, seeming to have arisen from the spherical
and ovoid forms by a process of lengthening and thinning. Of these elon-
gated forms, many appear hooked, or in some cases vacuolate, at the ends.
Mingled with these thread-like structures are others which appear circular,
as if a thread had formed a ring, or possibly a globular form had changed in
appearance so that it resembles a hollow sphere. At times, chains of small
granules are seen, probably due to the breaking up of a filament. Figure I
shows the large number and greatly varied shapes of the mitochondria in
Passing outward from the region of elongated cells in the plerome and
entering the periblem, the cells are found crowded with mitochondria,
mostly spherical in shape. It appears as if the mitochondria do not, in
general, elongate in this region, though they increase in size, approximately
to the same extent in all directions. As will be shown later, these enlarged
bodies of the periblem are in reality not mitochondria, but plastids, though
they stain in exactly the same manner with the haematoxylin. Whether
there are any plastids present in the plerome region as well, I am not as yet
prepared to say. Figure 2 shows a cell from the periblem in mitosis, drawn
to the same scale as figure 1, namely, nine hundred diameters. Figure 3
shows a similar cell enlarged to twice the size.
As may be seen from these drawings, the mitotic figure is shown very
much as it appears in the usual method of fixation, with the exceptions to
be noted. The spindle fibers are but faintly shown, if visible at all, although
the general outline of the spindle appears as it normally does. The chro-
mosomes are rather attenuated, though they sometimes show a shadowy
outline of surrounding material, as if only the central part of their structure
had been stained. The difference between this method of fixation and those
methods designed primarily for showing nuclear structure, is much more
marked in the resting cell. Here the nucleus appears, in general, more
uniformly granular and less reticulate than it does in preparations fixed,
say, in Flemming's strong fluid. The nucleole also is different, appearing
much larger than we are accustomed to see it in fixed material. In short,
the mitochondrial methods of fixation do not seem to alter the appearance
of the protoplast so much as do the usual types of fixation, since with the
mitchondrial methods the structure appears very much as it is described by
Lewis and Lewis ('15) and by Lewis and Robertson ('16), in their obser-
vations upon living tissue cells.
The root-tips from which the above described preparations were made
were grown in the laboratory during the coldest part of the winter and not
under constant temperature conditions. To this I attribute the difference
between the granular content of the cytoplasm in this material and in the
preparations next to be described.
Root-tips of the same variety of corn, grown later in the season and under
224 W. C. TWISS
more favorable conditions of temperature and light, prepared and stained
by the Benda method, show a much greater proportion of the enlarged
vesicular bodies which stain like mitochondria. In these preparations,
however, there is very good evidence that these bodies are in reality plastids,
since they show a lighter colored internal portion, evidently consisting of
starch. In regions apart from the meristem these bodies are very abundant.
In the plerome (fig. 4) the filamentous mitochondria often appear swollen
at the ends or sometimes in other portions, while the plastids, ovoid or
spherical in shape, may contain from one to several starch grains each as
shown in figure 5.
In the periblem, on the other hand, there are in the intermediate regions
of the tip, ovoid, spherical, or irregular masses of a much more solid appear-
ance, in general, but often showing a number of discrete spherical granules
in their interior. Numerous smaller spherical or ovoid bodies are also
present, scattered about through the cytoplasm, which are dark blue in
color, taking the stain exactly as do the plastids and the filamentous mito-
These preparations, stained and fixed according to the Benda method,-
as previously stated, show the nucleus finely granular in appearance and
of an old-rose color, as dark, relatively, as shown in the drawings, figures
4 and 5. The nucleole is apparently of a denser consistency and stains a
darker shade of the same color. The cytoplasm is very well preserved and
stains somewhat lighter than the nuclear material.
A slide of the above described material was freed from paraffin with
xylol, bleached for a few minutes in hydrogen peroxide, and treated with
potassium iodide-iodine solution, with the object of testing for starch.
Under this treatment the bodies which have been referred to as plastids
show a bluish color in their interior, but hardly pronounced enough to be
considered a convincing demonstration of the presence of starch. A sub-
sequent treatment of the slide with a solution of iodine in chloral hydrate,
however, gave better results, differentiating the bodies so that they appear
as plastids with included starch grains, as indicated by the blue color of the
Next, a similar slide from the Benda fixation was stained with the
Flemming three-color process, the red being left decidedly strong. The
mitochondria, both granular and filamentous, are now strongly stained by
the safranin, while the plastids are colored blue. In the intermediate regions
of the tip, in the periblem area, the plastids are rather lightly stained, and
within each of them there are a varying number of spherical granules which
are stained by the safranin in the same manner as the mitochondria, except
that they are, generally, a brighter red. In figure 6, a, b, c, d, and e, a
number of these plastids are shown with their included red-staining granules.
As one leaves the intermediate regions of the tip and proceeds toward the
proximal portion, the red bodies within the plastids gradually lose their
A STUDY OF PLASTIDS AND MITOCHONDRIA 225
sharply defined appearance and bright color, becoming gradually dimmer.
Still farther back in the tip the red color disappears entirely, the plastids
appearing more or less opaque or more uniformly blue in color, as shown in
figure 6, g, h, and i.
It has not been possible to determine, so far, how this association of the
bright red granules with the light blue plastids comes about, though it might
be imagined, from observations I have made, such as the appearance of
two darkly staining red granules at the ends of a light blue ellipsoid, that
the mitochondria are surrounded by, or become surrounded by, a substance
which makes up the body of the plastid ; that they divide within this plastid
substance and afterwards produce the starch grains within it, or themselves
become changed into starch. This appearance suggests a relation to starch
formation similar to that of the pyrenoid, as described by McAllister ('14).
In other cases, however, bodies were observed which are made up of a
dark red peripheral layer surrounding a light blue center. Both the latter
structures and the ellipsoids occur in the intermediate regions of the peri-
blem, between the red-staining mitochondria and the blue plastids with
their red, granular inclusions. I wish to emphasize the fact that both the
mitochondria and the plastids are exceedingly numerous in the regions
indicated and that the staining reactions and the differentiation of the bodies
described as occurring in the plastids are very definite. In many cells of
the periblem, in the intermediate region of the tip, the nucleus is practically
surrounded by a number of large plastids containing red-staining granules*
while the cells nearer the tip are crowded with mitochondria which also
color strongly with the safranin. Nevertheless, while the existence of
these bodies is clearly demonstrable, I do not wish to imply that the seriation
to prove their inter-relations is equally evident.
The situation in connection with the cytoplasmic inclusions of the
liverworts and of the Bryophyta in general, appears to be in special need of
investigation, not only on account of the fact that a perusal of the literature
shows a considerable difference of opinion as to the real nature of the various
bodies in question, but also on account of the very great interest attached to
the group by virtue of its intermediate position in relation to the flowering
plants on the one hand, and to the algae on the other.
While the interest centers mainly in the plastids and their genetic
relations, the oil bodies of the liverworts have received considerable atten-
tion from investigators, with no very definite results as far as their real
nature and origin is concerned. Pfeffer C74) is credited with having made
the first really fundamental and comprehensive study of the oil bodies.
In his opinion they are formed by the aggregation of a large number of
small oil droplets which are already visible in the very young cells. While
he, at first, maintained that the bodies originate in the cell sap, he later
226 W. C. TWISS
agreed that it might be possible that they come from the cytoplasm, but
that they finally lie in the vacuole. He believed the principal constituent
of the bodies to be a fatty oil, since their contents dissolve in alcohol,
benzol, ether, etc. In addition to the fatty oil, some other material was
found to be present, appearing as a residue after the solution of the oil.
The membrane which he observed surrounding the bodies after they had
been stained with iodine and with cochineal, was apparently composed of
some protein material, insoluble in dilute acid and in alkalies. Since the
bodies were unchanged after a three-months' cultivation of the plants in
darkness, and since they were still present, in such cases, in the very young
cells, just as in the plants which had grown in the light, he concluded that
they have no significance in nutrition and that they are merely products of
Wakker ('88) included the oil bodies of the liverworts under the "elaio-
plasts," as he had named the oil-producing bodies which he had demon-
strated in many phanerogams. Although these bodies appear, in life, to
lie in the cell sap, Wakker showed by abnormal plasmolyosis that they, in
reality, lie in the cytoplasm. He believed them analogous to leucoplasts
and chloroplasts, holding that they multiply by division and are distributed
to the daughter cells in mitosis.
Von Kiister ( '94) believes that the oil bodies are formed from a protein
"stroma" and that the apparent membrane seen in fixed material is an
artefact. Since he was not able to see the membrane in living material,
even with the strongest magnification, he considered it a precipitation
membrane, formed by the interaction of the oil and the substance of the
stroma. He showed that the membranes were not visible in material which
had been fixed in osmic acid and stained with gentian violet. He also
showed that a double membrane could be formed by the use of dilute
alcohol, followed by strong. In regard to the nature of the bodies, he
believed with Pfeffer that they are excretion products. He did not believe
that the oil bodies undergo division and are handed down from cell to cell,
but thought that they are newly formed in each cell.
Garjeanne ('03) believes that it is possible to show that the oil bodies
are in reality merely vacuoles filled with oil which is secreted from their
walls; that they lie in a half-fluid transition substance; that they increase
by division, and that the membrane is an artefact. He admits, however,
that the picric acid which he used in his demonstrations acts very rapidly,
so that observations upon young cells must be made within one minute
after the application of the acid, before disorganization of the cell contents
sets in. He compares the oil bodies to the leucoplasts in their origin from
Anlagen, which he believes to be vacuoles in the case of the leucoplasts also.
After being fully formed, the oil bodies, he says, are no longer capable of
division, remaining thereafter unchanged. In addition to the vacuoles, or
Anlagen, of the oil bodies, he describes other minute structures which are
A STUDY OF PLASTIDS AND MITOCHONDRIA 227
similar to the young stages of the oil bodies but which differ from them in
their chemical properties. Since, in addition to the Anlagen of the oil
bodies and to these structures which are similar to them, Garjeanne men-
tions also the Anlagen of the chloroplasts, it would seem that he believes
that there are present in the cells of some liverworts, at least three varieties
of specifically different granules.
Rivett ('18), in an account of certain observations upon Alicularia
scalaris, finds that the results of staining or fixing the entire leaves with
2 percent osmic acid "confirm the view that the oil is secreted in vacuoles."
This author also finds certain refractive granules present in the cells of both
the growing point and the older leaves, which differ in their chemical reac-
tions from the young oil bodies, apparently agreeing with the observations
of Garjeanne in this respect. In the meristematic regions a "chondriome-
like structure" was observed, but no evidence was found "that the chon-
driosomes were either transformed directly into plastids by a secretion
within their own substance, or that they are the instigators of secretory
action on the part of the protoplasm." No evidence was found that the
refractive granules were chondriosomes, "since their appearance in the
stained mature cells is quite different from that of the chondriome of the
actively dividing cells."
As already noted, mitochondria have also been described in the liver-
worts by Scherrer ('13) and by Mottier, ('18), neither of whom was able
to find any connection between these bodies and the chloroplasts. Scherrer
made a special study of Anthoceros, a form which possesses the greatest
interest since it has a "pyrenoid' in some of its chloroplasts, suggesting a
close relationship with the algae in respect to its method of starch formation.
The pyrenoid of Anthoceros has been described by McAllister ('14),
who finds that it consists of from 20 to 300 minute lenticular bodies, which
lie near the center of the chloroplast and which stain bright red with safranin.
McAllister states that there can be no doubt that these bodies are trans-
formed directly into starch, since "there is a gradual change of the color
reaction, from the brilliant red of the pyrenoid bodies to the blue of the
starch grains." On the other hand, he says that, in the cells of the arches-
porium, in the spore mother cells, and in the assimilative cells of the sporo-
phyte, starch is formed without the intermediary action of a pyrenoid —
apparently arising de novo in the chloroplasts.
The observations of Davis ('99) agree with those of McAllister in this
respect, since be states that the first clear indication of the chloroplast in
the spore mother cells of Anthoceros is the sharp staining of the starch
grains — purple with the gentian violet.
McAllister states, further, that there is no doubt that if the Anlagen of
these bodies (the starch grains of the spore mother cells) are present in the
plastids of the archesporial cells, they are too minute to be distinguished with
the highest magnifications. This, however, does not necessarily follow,
228 W. C. TWISS
since neither McAllister nor Davis reports having tried the mitochondrial
methods of fixation.
Although Anthoceros, in general, has but one chloroplast in each cell,
Campbell ('o6) has described a species from Jamaica which has several
chloroplast^ — as many as eight in the cells of the inner tissue — so that the
connection of Anthoceros with other liverworts, in this respect, is not so
remote as might at first appear. All in all, Anthoceros is obviously a most
interesting form and one upon which considerably more work is necessary.
Portions of the thallus of Preissia commutata, upon which the gamete-
bearing discs were beginning to appear, were fixed according to Benda's
formula, imbedded, and cut 5 microns in thickness. The smallest disc
studied was about one millimeter in diameter. Figure 7 represents a portion
of such a disc, as it appeared when stained with the Benda method, and
with a magnification of three hundred eighty-four diameters. The darker
cells in this section, three of which are shown in figure 7, present the same
relatively dark appearance in the unstained, unbleached preparations.
They are filled with a dense mass of thin-walled, spherical bodies which
stain darkly with osmic acid as well as with the mitochondrial stains.
Treatment with a preparation of alcannin shows the periphery of these
cells made up of an alveolar substance, staining purplish gray, while the
central portion contains a mass of material which stains a dark red. These
central masses are the "oil bodies" of Pfeffer and others, or the "elaioplasts"
In this section there are also differentiated two other sorts of bodies.
The smaller, more uniform variety, which may be seen occupying the periph-
ery of the cells, is apparently of a fatty nature, since they are somewhat
darkened in the unbleached cells. They appear granular and plastic,
being flattened more or less along the cell walls. There appears to be no
difference between the periphery and the interior of these bodies, since no
bounding membrane nor any lighter-colored area in the interior can be
made out in the stained slides. From the larger ones, two microns or more
in diameter, of which there are usually a larger number of about the same
dimensions in these cells, they seem to grade down to extremely minute
The other bodies vary much more in size, there being no two of any one
size in the cell. They include, doubtless, the bodies described as oil drop-
lets by Pfeffer. Figures 8, 9, and 10 show a few of the cells taken from the
same group as figure 7, but more highly magnified. The more uniformly
colored bodies in these cells, mostly seen in side view around the periphery
of the cell, belong to the first class, while the more rounded ones, with dark
borders, belong to the second class. Of the latter, the larger ones may ap-
pear to be in a state of division or fusion, a number, of varying sizes, often
A STUDY OF PLASTIDS AND MITOCHONDRIA 229
appearing in groups; figure 11, plate II, shows a peripheral cell from a
young disc containing the two sorts of bodies of the second class.
Returning to figure 7, as one proceeds from the periphery of the young
disc toward its center, the bodies of the second class appear, on the whole,
smaller. There seems to be a gradual decrease in their size correlated with
an increase in their number, up to a depth of about two cell layers below
the areolae. Here, bodies of this class begin to decrease in number, indi-
cating a diminution in the amount of oil in the cells, while bodies containing
from one to several starch grains begin to be seen: figures 8 and 9.
This seriation, suggesting that at least some of the bodies are plastids,
is better shown in figures 13 and 14, taken together, which were drawn
from a somewhat older disc than that from which figure 7 was taken.
Figure 14 shows two cells from still deeper within the disc, as compared with
figure 13, from a central lenticular area in which all the cells are of this
type and packed with storage starch. These plastids, for such they appear
to be, are stuffed with starch and now appear merely as enveloping films,
enclosing and separating the starch grains. The character of these starch
grains, which show a definite hilum when stained in certain ways, as well as
their general appearance and distribution, would indicate that they are, as
already suggested, storage starch and that the grains are very likely strati-
fied. It was also found on staining such a section as that shown in figure 12
with iodine, that the starch reaction was given by all the bodies of this
character, even to some of the smaller ones in the peripheral layer of the
disc. Figure 15 represents a series of stages in outline as they are seen in
the development of starch in this disc.
In the chloroplasts of the thallus, starch is also present in large quan-
tities, and toward the interior of the thallus there is a region in which the
cells are moderately full of swollen plastids, each containing a number of
plump starch grains, not at all lenticular in appearance as they are so often
figured. Lenticular-shaped starch grains are found, however, in the chloro-
plasts of cells at and near the periphery, especially in the vicinity of the
growing point of the thallus. In the cells of the disc which contain starch,
as well as in those of the thallus, the smaller, more plastic, and darker-
staining bodies are still seen, arranged around the periphery. The largest
of these should be the young chloroplasts.
In the apical cell of the thallus and in its immediate vicinity, bodies may
be seen which I take to be the same as those already described from the
peripheral cells of the young disc, though they are much smaller in size.
In this region, also, and especially in the filamentous growths therefrom,
mitochondria of various shapes appear, very much as described by Mottier
('18) for Marchantia.
It was at one time the accepted belief among botanists that chloroplasts
arise de novo in the cytoplasm. This is definitely stated to be the case by
230 W. C. TWISS
Sachs, in his Text-book of Botany, in 1882, where the process is compared
to that of so-called free-cell formation. Since the work of Schimper,
afterwards confirmed by that of A. Meyer, the opinion has become general
that the three kinds of plastids found in plant cells, namely, leuco-, chloro-,
and chromoplasts, are derived from minute, undifferentiated plastids which
are sui generis structures of the cytoplasm. These chromatophores, as
they are often called, were described, when seen in the living cell, as small,
colorless, highly refractive bodies, recognizable in the egg and also in the
embryonic cells. In older cells they have been said to retain the same
appearance in some cases, while in others they become differentiated into
leuco-, chloro-, and chromoplasts.
Schimper and Meyer believed that the undifferentiated plastids multiply
by division and are handed down from generation to generation — that they
have an individual existence in the cells. Considerable difficulty, however,
was encountered by them in their attempts to demonstrate the presence of
the plastids in the egg } owing to the fact that they were not easily seen in
the living cells, and, as was admitted, they were difficult to stain at that
stage. As Guilliermond expresses it, "that part of their theory remained
very hypothetical.' '
When the mitochondria were demonstrated, by means of a special
technique, their study was first taken up by the zoologists, as has been shown,
and special functions in the cell metabolism were imputed to them by
Meves and others. Pensa is credited with having made the first observa-
tions tending to show that the mitochondria of plants may, possibly, be
transformed plastids. This idea, developed by Lewitsky and Pensa and
supported by numerous observations which have already been noted, was
taken up by Guilliermond, who has attempted particularly to harmonize
the functions of the mi ochondria of plant cells with the theories concerning
those of the mitochondria of animal cells as postulated by Meves, Dues-
berg, Regaud, Dubreuil, and others. He has confirmed the observations
of Lewitsky and of Pensa by work upon a number of plants, including the
seedling of barley. Here the mitochondria, followed from the meristem
toward the green tip of the plumule, are shown as filamentous at first,
followed by shorter and thicker forms which are sometimes dumb-bell
shaped. From the appearance in succeeding cells of bodies which have the
appearance of the separated halves of the " dumb-bells/' he believes that
the latter divide. These bodies are followed by more rounded forms with
a light center and a darker border. Finally, in the tip of the plumule, the
mature chloroplasts are seen. While this series is considered by Guillier-
mond a very convincing proof of the mitochondrial origin of the chloroplasts,
it is open to the objection that there seems to be no way of demonstrating
that the Anlagen of the plastids are actually mitochondria and not merely
On the other hand, the attempts of Rudolph, Sapehin, Mottier, and
A STUDY OF PLASTIDS AND MITOCHONDRIA 23 1
others to substantiate their contention that the Anlagen of the plastids are
different from the mitochondria fail for the same reason — they are unable
to demonstrate such a difference. Guilliermond ('14) claims that he is, in
reality, upholding the Schimper-Meyer theory in that he is bridging a gap
which the latter, with their cruder technique, were unable to fill. Schmidt
('12) also maintains that the work of Guilliermond and others confirms the
Schimper-Meyer theory, but for a very different reason : they have simply
been demonstrating the earlier stages of the plastids, according to Schmidt.
Lewitsky, however, claims to have shown that there are no other Anlagen
of the chloroplasts than the mitochondria. His findings, with respect to
Asparagus officinalis, which have already been referred to, are specifically
as follows: a stem-tip of Asparagus officinalis was fixed with alcohol, 3
parts, and acetic acid, 1 part. This was stained with iron-alum-haema-
toxylin and light green. In the third and fourth cell-layers from above,
where, in the preparations fixed by the Benda method, the somewhat large
" chroma tophore-dumb-bells ,, were found, these were no longer to be seen;
only the usual " Plasmagervst" was present. Since the mitochondria are
destroyed by acetic acid and alcohol, and since all of the Anlagen, including
the dumb-bell forms, disappear with the mi ochondria under fixation with a
combination of the above named reagents, these facts are taken as conclusive
evidence of the identity of the mitochondria with the Anlagen of the plastids.
My own observations may be briefly summarized as follows :
1 . As to size, an unbroken series of bodies, from mitochondria to plastids,
can be traced in the root-tip cells of Zea Mays from the embryonic region
backward. In Preissia this seriation is not so obvious.
2. The contention that such definitely staining bodies as the mitochon-
dria exist and are normal constituents of the cytoplasm can hardly be
3. The evidence for the division of the mitochondria as well as that for
their functions in heredity seems to me to be inadequate.
4. The further fundamental question as to the relation of the mitochon-
dria to the remainder of the cytoplasm and the nature of the material in
which they are imbedded, has not been cleared up.
5. Red-staining bodies are present in the plastids of corn, and, in some
cases, in those of Preissia also.
Beer, R. 1905. On the development of the pollen grain and anther of some Onagraceae.
Beih. Bot. Centralbl. 19: 286-311.
Campbell, D. H. 1906. Multiple chromatophores in Anthoceros. Annals of Botany 20:
Cavers, F. 1914. Chondriosomes (mitochondria) and their significance. New Phytol.
13: 96-106; 170-180.
Cowdry, N. H. 19 17. A comparison of mitochondria in plant and animal cells. Biol.
Bull. 33: 196-228.
232 W. C. TWISS
Davis, B. M. 1899. The spore mother cell of Anthoceros. Bot. Gaz. 28: 89-109.
Derschau, M. v. 1907. Uber Analogieen pflanzlicher und tierischer Zellstrukturen.
Beih. Bot. Centralbl. 22: 167-190.
. 191 1. Ueber Kernbriicken und Kernsubstanz in pflanzlichen Zellen. Arch. Zellf.
D obeli, C. C. 1909. Chromidia and the binuclearity hypothesis. Quart. Journ. Micr.
Dubreuil, G. 1913. Le chondriome et le dispositif de l'activite secretaire. Arch. Anat.
Micros. 15: 53-I5I-
Duesberg, J. 19 10. Les chondriosomes des cellules embryonnaires du poulet et leur
role dans la genese des myofibrilles. Arch. Zellf. 4: 602-671.
Duesberg, J., and Hoven, H. 1910. Observations sur la structure du protoplasme dls
cellules vegetales. Anat. Anz. 36: 96-100.
Faure'-Fremiet, E. 1908. La structure des matieres vivantes. Bull. Soc. Zool. France
. 1910. Etude sur les mitochondries des Protozoaires et des cellules sexuelles. 11:
457-648. Arch. Anat. Micros.
Forenbacher, A. 191 1. Die Chondriosomen als Chromatophorenbildner. Ber. Deutsch.
Bot. Ges. 29: 648-660.
Garjeanne, A. J. M. 1903. Die Olkorper der Jungermanniales. Flora 92: 457-482.
Goldschmidt, R. 1904. Der Chromidialapparat lebhaft funktionierender Gewebezellen.
Biol. Centralbl. 24: 241-251.
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Acad. Sci. (Paris) 153: 199-201.
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chondries. Rev. Gen. Bot. 26: 129-149; 183-208.
Hertwig, R. 1899. Ueber Encystierung und Kernvermehrung bei Arcella vulgaris.
Festscrift fur Kupffer, pp. 367-382. Jena.
Ktister, v. 1894. Die Olkorper der Lebermoose. Basel.
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in tissue cultures. Amer. Journ. Anat. 17: 339~4 01 -
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observed by the tissue culture method. Biol. Bull. 30: 99-124.
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Bot. Ges. 28: 538-546.
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canadensis Rich. Ber. Deutsch. Bot. Ges. 29: 697-703.
Lowschin, A. M. 19 13. "Myelinformen" und Chondriosomen. Ber. Deutsch. Bot
Ges. 31: 203-209.
Lundegard, H. 1910. Ein Beitrag zur Kritik zweier Vererbungshypothesen. Jahrb.
Wiss. Bot. 48: 285-378.
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Pflanzenzellen. Ber. Deutsch. Bot. Ges. 22: 284-286.
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A STUDY OF PLASTIDS AND MITOCHONDRIA 233
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EXPLANATION OF PLATES XXXIII-XXXIV
All figures were drawn with the aid of the camera lucida, with a Leitz No. 3 ocular and
Leitz 1/16 in. oil immersion lens, with the following exceptions: In figure 7 a Leitz No. 6
objective was used; in figures 13 and 14, a Spencer 1/12 in. oil immersion lens.
Fig. I. Cell from plerome of root-tip of corn, showing mitochondria. X 900.
Fig. 2. Cell from periblem of same preparation, showing mitotic figure and ovoid
and spherical mitochondria. X 900.
Fig. 3. Cell from same region showing similar structures. X 1800. All of above
from Regaud 's fixation and haematoxylin stain.
Fig. 4. Cell from plerome of corn root-tip, from plant grown under more favorable
conditions. Benda's method of fixation and staining. Vesicular structures show light
blue interior. X 900.
Fig. 5. Two cells from periblem of same preparation. Mitochondria ovoid or spher-
ical. X 900.
Fig. 6. Series of plastids from same preparation, stained with Flemming's tri-color
process. First six in series have red-staining granular inclusions; g, h, and i are stained
blue. Iodine test shows starch grains in latter. (Fig. 6 on Plate XXXIV.)
Fig. 7. Section from young disc of Preissia, showing distribution of the granular
material in the cells and in the thallus as a whole. X 385*
Figs. 8, 9, and 10. Cells taken from same section, showing plastids, etc., on larger
scale. X 900.
234 w - c - twiss
Fig. i I . Cell from periphery of young disc of Preissia. Benda's method (unbleached) .
Large bodies with dark borders, violet; smaller, uniformly-colored structures, brown.
Fig. 12. Cell from same region, showing only larger bodies. X 1800.
Fig. 13. Portion of older disc, showing development of plastids, with large grains of
storage starch. Iodine stain, with Benda's fixation. X 800.
Fig. 14. Two cells from central part of same disc, showing fully developed grains.
Fig. 15. Series of developing starch grains, from near the periphery of the disc to its
center. X 900. (Fig. 15 on Plate XXXIII.)
Fig. 16. Cell from thallus of Preissia. Benda's method (unbleached). Large
"chloro-leucoplasts" light blue, with dark violet peripheries and filled with starch grains.
Smaller bodies, dark brown. X 900.
American Journal of Botany.
Volume VI, Plate XXXIII.
I .-..■ • •• •■•,„••
American Journal of Botany
Volume VI, Platb XXXIV.
Twiss: A study of plastids and mitochondria